PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) has the highest mortality rate of all cancers, is rapidly becoming the second leading cause of cancer deaths in the U.S., and is estimated to cost the health care system $2.4 billion each year. Patients with PDAC have a 5-year survival rate of 7% and that number drops to 2% for individuals with metastatic disease. Unfortunately, there has been little progress in shifting patient outcome over the past 40 years, highlighting the need for innovative approaches to define the molecular pathways that influence PDAC development. PDAC can be initiated in exocrine epithelial cells that acquire an activating mutation in the KRAS protooncogene, causing the cells to transition to pancreatic intraepithelial neoplasia (PanIN) lesions that can progress into PDAC. A key transcriptional response to KRAS activity is increased expression of the physiological activator of the coagulation system, Tissue Factor (TF). TF mediates conversion of prothrombin to the active protease thrombin that in turn promotes extensive fibrin deposits within the tumor microenvironment (TME) and activates G-protein coupled protease-activated receptors (PARs) on target cells, including PDAC tumor cells, tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs). The scientific premise is based on a known relationship between pancreatic cancer and high-level blood coagulation system activity, but is unique in that it will define newly discovered mechanisms of crosstalk by which specific coagulation factors in the TME promote PDAC pathogenesis. Our central hypothesis is that thrombin drives early PanIN development as well as advanced PDAC tumor growth and metastasis by mechanisms linked to both fibrin matrix deposition in the TME and PAR-1 signaling on tumor and TME cells promoting a feed forward pathway of PDAC disease. This hypothesis will be tested by utilizing a combination of unique genetically engineered mouse models, cutting-edge pharmaceutical reagents, and patient-derived PDAC tumor cells to pursue three Specific Aims - (1) determine how the thrombin-fibrin axis contributes to early PanIN development and late stage PDAC tumor growth and metastasis; (2) determine the individual mechanisms by which thrombin receptor PAR-1 signaling on tumor cells, TAMs and CAFs exacerbates distinct aspects of PDAC pathogenesis; and (3) determine the efficacy of pharmacological fibrin and PAR-1 inhibition in halting the progression of established mouse PDAC and human patient-derived xenograft tumors. These studies are significant because they will (i) be the first to define the impact of thrombin throughout PDAC disease; (ii) identify specific mechanisms of thrombin-dependent PDAC disease progression; and (iii) provide novel opportunities to target PDAC tumors using specific state-of-the-art inhibitors of fibrin matrix and PAR-1 signaling pathways. The discoveries made will greatly advance the understanding of mechanisms underlying PDAC disease and are expected to highlight novel therapeutic opportunities for developing improved treatment options - a long-term goal with the greatest benefit to patient outcome.